The laboratory focus of the Molecular Therapeutics Section this past year has been on increasing our understanding of non-P-glycoprotein mechanisms of drug resistance. We have examined a novel mechanism of drug efflux and resistance in human breast cancer cells; the role of growth factor signaling in drug resistant breast cancer cells; and recently have begun investigations into the intrinsic mechanisms of resistance in renal cell carcinomas. The doxorubicin-resistant breast cancer subline we have characterized appears to contain a new ATP-dependent membrane transporter which has particular activity for mitoxantrone, daunorubicin, and doxorubicin, but which is not P glycoprotein (Pgp) or the multidrug resistance associated protein (MRP). Efflux can be reversed by a compound we have obtained from Wyeth-Ayerst, but cannot be reversed by standard P-glycoprotein antagonists. A major effort is underway to identify the protein, and to clone the gene encoding the protein. Growth factor signaling may be involved in the response of breast cancer cells to drug exposure. Resistant cell lines demonstrate overexpression of growth factors or their receptors, but display inhibition of components of the signaling pathway. Efforts are underway to identify the relationship of this downregulation of signaling to the reduced proliferation observed in the cells. Recently, we have begun to focus on intrinsic resistance in renal cell carcinoma. Biopsies performed on tumor samples from patients with renal cell carcinoma enrolled on the PSC 833 and vinblastine trial demonstrated low levels of expression of mdr-1. Studies in paired renal cell carcinoma cell lines derived from primary and metastatic lesions indicate that the metastatic lesions have lower levels of mdr-1 than the primary lesions. Studies are ongoing which suggest the presence of non-Pgp, non-MRP mechanisms of drug efflux in renal cell carcinoma. We plan to evaluate the contribution of these drug efflux mechanisms to the intrinsic resistance so characteristic of this malignancy, and subsequently to examine intracellular mechanisms of resistance as well. The clinical focus for the Molecular Therapeutics Section continues to be the Phase I clinical trials testing P-glycoprotein reversal. A Phase I trial to determine the MTD for the cyclosporine analogue, PSC 833, in combination with vinblastine is complete. A Phase I trial combining PSC 833 with paclitaxel is ongoing. We plan to open a Phase II trial of PSC 833 in combination with infusional vinblastine in the coming year. That trial is spurred by responses noted in the Phase I trial. Laboratory support for the clinical trials includes analysis of biopsies from patients on study for mdr-1 and MRP expression; and processing of pharmacokinetic samples. Finally, we have continued our interactions with the Developmental Therapeutics Program and the NCI drug screen in identifying P-glycoprotein substrates and antagonists; EGF and ErbB2 inhibitors; MRP substrates; and most recently, compounds highly active in renal cell carcinoma cell lines.